Rapid Microbial Testing

Testing for the presence of microbes in an aseptic environment used to be a process that took weeks. Now, methods for rapid microbial testing can be used to detect the presence of mircoorganisms in the environment or in a finished drug product in hours or days. In this technical forum, experts describe different methods of rapid microbial testing and their applications. Contributors to the forum are: Anne Baumstummler, senior scientist; Renaud Chollet, head of biotechnologies and reagents; Hervé Meder, R&D project manager; Céline Rofel, cell culture and molecular biology technician; Adrien Venchiarutti, engineer; and Sébastien Ribault, PhD, director development & bioproduction, all at Millipore SAS; and Ruth Eden, PhD, president of BioLumix.

Traditional testing methods for microbial contamination of mammalian cell culture require several days to detect contamination. The presence of microorganisms is typically assessed by membrane filtration and incubation on solid media or inoculation into liquid media. These culture-based methods rely on microorganisms growing and yielding visible colonies or turbidity. Time-to-result is measured in days for standard bioburden testing (1). The microcolonies fluorescent staining method (MFSM) described in this study is a robust, simple, and rapid method enabling the detection of microbial contaminants. It can be applied to cell culture samples with time-to-results that are two to five times shorter than traditional microbiology. The nondestructive feature makes it compatible with downstream identification of contaminants.

The need for speed. Many technologies and detection systems such as enzyme-linked immunosorbent assays, impedimetry, bioluminescence, flow cytometry, and polymerase chain reaction arose in the field of rapid detection during the two past decades (2–4). Fluorescent dyes have been routinely used in methods such as the direct epifluorescent filter technique (DEFT) to monitor microbial contamination (5). This technique involves the capture of microorganisms on the surface of polycarbonate membrane filters, staining, and visualization using epifluorescence microscopy. The sensitivity is directly linked to the volume filtered and the number of fields observed. A limitation of this approach is the inability to differentiate fluorescent microorganisms from autofluorescent particles. Automated and semi-automated systems have been developed to increase this discrimination and improve accuracy (6–8).

An alternative is to combine the principles of epifluorescence microscopy and flow cytometry (9). Microorganisms retained on a polycarbonate filter are fluorescently labeled and automatically counted by a laser-scanning device that makes the differentiation between fluorescent microorganisms and particles (10). Several authors have reported the use of this solid-phase cytometry technology for bioburden testing of mammalian cell culture processes (1). Results are typically obtained within five hours of sample preparation (6). One drawback of such direct detection methods is the inability to discriminate between culturable and nonculturable organisms (11). In addition, many fluorescent-labeling techniques have an impact on the viability of colony formation, thereby making it impossible to identify organisms that are detected. This is a challenge when conducting investigations or evaluating the severity of a contamination.

Figure 1

MFSM is a method that combines the widely used membrane filtration method with a fluorescence-based staining for the rapid detection of contaminants in filterable samples using microcolony formation. MFSM is based on a nondestructive fluorescent labeling of viable and culturable microorganisms present on cellulose membranes (MilliFlex Quantum, EMD Millipore; see Figure 1). The procedure consists of filtration of the sample, a short incubation on media to yield microcolonies, staining of microorganisms, and counting of fluorescent microcolonies with a light-emitting diode (LED) system. The microorganisms are labeled directly on the filter with a nonfluorescent substrate that is cleaved by intracellular microbial enzymes. Only metabolically active microorganisms with membrane integrity that retain the fluorescent product are stained. Because the staining solution is nondestructive, the method is compatible with standard identification methods.

The objective of this study was to compare MFSM with traditional epifluorescence microscopy for its ability to rapidly detect microbial contaminants in mammalian cell culture.